Far-Red Fluorescent Senescence-Associated β-Galactosidase Probe for Identification and Enrichment of Senescent Tumor Cells by Flow Cytometry

Summary

A protocol for fluorescent, flow cytometric quantification of senescent cancer cells induced by chemotherapy drugs in cell culture or in murine tumor models is presented. Optional procedures include co-immunostaining, sample fixation to facilitate large batch or time point analysis, and the enrichment of viable senescent cells by flow cytometric sorting.

Abstract

Cellular senescence is a state of proliferative arrest induced by biological damage that normally accrues over years in aging cells but may also emerge rapidly in tumor cells as a response to damage induced by various cancer treatments. Tumor cell senescence is generally considered undesirable, as senescent cells become resistant to death and block tumor remission while exacerbating tumor malignancy and treatment resistance. Therefore, the identification of senescent tumor cells is of ongoing interest to the cancer research community. Various senescence assays exist, many based on the activity of the well-known senescence marker, senescence-associated beta-galactosidase (SA-β-Gal).

Typically, the SA-β-Gal assay is performed using a chromogenic substrate (X-Gal) on fixed cells, with the slow and subjective enumeration of "blue" senescent cells by light microscopy. Improved assays using cell-permeant, fluorescent SA-β-Gal substrates, including C₁₂-FDG (green) and DDAO-Galactoside (DDAOG; far-red), have enabled the analysis of living cells and allowed the use of high-throughput fluorescent analysis platforms, including flow cytometers. C₁₂-FDG is a well-documented probe for SA-β-Gal, but its green fluorescent emission overlaps with intrinsic cellular autofluorescence (AF) that arises during senescence due to the accumulation of lipofuscin aggregates. By utilizing the far-red SA-β-Gal probe DDAOG, green cellular autofluorescence can be used as a secondary parameter to confirm senescence, adding reliability to the assay. The remaining fluorescence channels can be used for cell viability staining or optional fluorescent immunolabeling.

Using flow cytometry, we demonstrate the use of DDAOG and lipofuscin autofluorescence as a dual-parameter assay for the identification of senescent tumor cells. Quantitation of the percentage of viable senescent cells is performed. If desired, an optional immunolabeling step may be included to evaluate cell surface antigens of interest. Identified senescent cells can also be flow cytometrically sorted and collected for downstream analysis. Collected senescent cells can be immediately lysed (e.g., for immunoassays or 'omics analysis) or further cultured.

Introduction

Senescent cells normally accumulate in organisms over years during normal biological aging but may also develop rapidly in tumor cells as a response to damage induced by various cancer treatments, including radiation and chemotherapy. Though no longer proliferating, therapy-induced senescent (TIS) tumor cells may contribute to treatment resistance and drive recurrence^1,2,3. Factors secreted by TIS cells can exacerbate tumor malignancy by promoting immune evasion or metastasis^4,5. TIS cells develop complex, context-specific phenotypes, altered metabolic profiles, and unique immune responses^6,7,8. Therefore, the identification and characterization of TIS tumor cells induced by various cancer treatment approaches is a topic of ongoing interest to the cancer research community.

To detect TIS tumor cells, conventional senescence assays are widely used, primarily based on detecting increased activity of the senescence marker enzyme, the lysosomal beta-galactosidase GLB1⁹. Detection at a near-neutral (rather than acidic) lysosomal pH allows for specific detection of senescence-associated beta-galactosidase (SA-β-Gal)¹⁰. A standard SA-β-Gal assay that has been used for several decades uses X-Gal (5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside), a blue chromogenic beta-galactosidase substrate, to detect SA-β-Gal in fixed cells by light microscopy¹¹. The X-Gal assay allows the qualitative visual confirmation of TIS utilizing commonly available reagents and laboratory equipment. A basic transmitted light microscope is the only instrumentation required to evaluate the presence of the blue chromogen. However, the X-Gal staining procedure can lack sensitivity, sometimes requiring more than 24 h for color to develop. Staining is followed by low-throughput, subjective scoring of individual senescent cells based on counting the cells exhibiting some level of intensity of the blue chromogen under a light microscope. As X-Gal is cell-impermeable, this assay requires solvent-fixed cells, which cannot be recovered for downstream analysis. When working with limited samples from animals or patients, this can be a major drawback.

Improved SA-β-Gal assays using cell-permeant, fluorescent enzyme substrates, including C₁₂-FDG (5-dodecanoylaminofluorescein Di-β-D-Galactopyranoside, green) and DDAOG (9H-(1,3-dichloro-9,9-dimethylacridin-2-one-7-yl) β-D-Galactopyranoside, far-red) have previously appeared in the literature^12,13,14,15. The chemical probe structure and optical characteristics of DDAOG are shown in Supplementary Figure S1. These cell-permeant probes permit the analysis of living (rather than fixed) cells, and fluorescent rather than chromogenic probes facilitate the use of rapid high-throughput fluorescent analysis platforms, including high-content screening instruments and flow cytometers. Sorting flow cytometers enable the recovery of enriched populations of living senescent cells from cell cultures or tumors for downstream analysis (e.g., western blotting, ELISA, or 'omics). Fluorescence analysis also provides a quantitative signal, allowing for more accurate determination of the percentage of senescent cells within a given sample. Additional fluorescent probes, including viability probes and fluorophore-labeled antibodies, can readily be added for multiplexed analysis of targets beyond SA-β-Gal.

Similar to DDAOG, C₁₂-FDG is a fluorescent probe for SA-β-Gal, but its green fluorescent emission overlaps with intrinsic cellular AF, which arises during senescence due to the accumulation of lipofuscin aggregates in cells¹⁶. By utilizing the far-red DDAOG probe, green cellular AF can be used as a secondary parameter to confirm senescence¹⁷. This improves assay reliability by using a second marker in addition to SA-β-Gal, which can often be unreliable as a single marker for senescence¹⁸. As the detection of endogenous AF in senescent cells is a label-free approach, it is a rapid and simple way to expand the specificity of our DDAOG-based assay.

In this protocol, we demonstrate the use of DDAOG and AF as a rapid, dual-parameter flow cytometry assay for the identification of viable TIS tumor cells from in vitro cultures or isolated from drug-treated tumors established in mice (Figure 1). The protocol uses fluorophores compatible with a wide range of standard commercial flow cytometry analyzers and sorters (Table 1). Quantitation of the percentage of viable senescent cells using standard flow cytometry analysis is enabled. If desired, an optional immunolabeling step may be performed to evaluate cell surface antigens of interest concurrently with senescence. Identified senescent cells can also be enriched using standard fluorescence-activated cell sorting (FACS) methodology.

Figure 1: Experimental workflow. A schematic summarizing key points of the DDAOG assay. (A) A TIS-inducing drug is added to mammalian cultured cells or administered to tumor-bearing mice. Time is then allowed for the onset of TIS: for cells, 4 days following treatment; for mice, 22 days total, with three treatments every 5 days plus 7 days recovery. Cells are harvested or tumors are dissociated into suspension. (B) Samples are treated with Baf to adjust lysosomal pH for detection of SA-β-Gal for 30 min; then, DDAOG probe is added for 60 min to detect SA-β-Gal. Samples are washed 2x in PBS, and a viability stain is briefly added (15 min). Optionally, samples can be stained with fluorescent antibodies in open fluorescence channels and/or fixed for later analysis. (C) Samples are analyzed using a standard flow cytometer. Viable cells are visualized in dot plots showing red DDAOG (indicating SA-β-Gal) versus green autofluorescence (lipofuscin). A gate to determine the percentage of TIS cells is established based on untreated control samples (not shown). If a sorting cytometer (FACS) is used, TIS cells can be collected and placed back into culture for further in vitro assays or lysed and processed for molecular biology assays. Abbreviations: DDAO = 9H-(1,3-dichloro-9,9-dimethylacridin-2-one); DDAOG = DDAO-Galactoside; TIS = therapy-induced senescence; FL-Ab = fluorophore-conjugated antibody; Baf = Bafilomycin A1; SA-β-Gal = senescence-associated beta-galactosidase; PBS = phosphate-buffered saline; FACS = fluorescence-activated cell sorting. Please click here to view a larger version of this figure.

Fluorophore	Detects	Ex/Em (nm)	Cytometer laser (nm)	Cytometer detector / bandpass filter (nm)
DDAOG	SA-β-Gal	645/6601	640	670 / 30
AF	Lipofuscin	< 600	488	525 / 50
CV450	Viability	408/450	405	450 / 50
PE	Antibody/surface marker	565/578	561	582 / 15

Table 1: Fluorophores and cytometer optical specifications. Cytometer specifications used in this protocol are listed for an instrument with a total of 4 lasers and 15 emission detectors. DDAOG detected at 645/660 nm is the form of the probe cleaved by SA-β-Gal¹. Uncleaved DDAOG can exhibit low level fluorescence at 460/610 nm but is removed by wash steps in the protocol. Abbreviations: DDAO = 9H-(1,3-dichloro-9,9-dimethylacridin-2-one); DDAOG = DDAO-Galactoside; AF = autofluorescence; PE = phycoerythrin; SA-β-Gal = senescence-associated beta-galactosidase.

Protocol

All animal work described was approved by the Institutional Animal Care and Use Committee at the University of Chicago.

1. Preparation and storage of stock solutions

NOTE: If cells will be flow-sorted, all solutions should be prepared using sterile techniques and filtered through a 0.22 µm filter device.

1. Prepare a stock solution of DDAO-Galactoside at 5 mg/mL in DMSO. Aliquot at 50 µL per tube (or volume desired). Store at −20 °C in the dark for up to 1 year.
2. Prepare a stock solution of Bafilomycin A1 at 1 mM in DMSO. Aliquot at 50 µL per tube (or volume desired). Store at −20 °C for up to 6 months.
3. Prepare a stock solution of Calcein Violet 450 AM at 1 mM in DMSO. Aliquot at 50 µL per tube (or volume desired). Store at −20 °C in the dark for up to 1 year.
4. For treatment of cultured cells in vitro, prepare 10 mM concentrated stock solutions of senescence-inducing agent(s) in the appropriate solvent, and sterilize using 0.2 µm syringe filters. Store at −20 °C or as directed by the manufacturer.
   NOTE: For delivery of senescence-inducing chemotherapy agents in vivo (to mice with established tumors), the agents should be USP grade and diluted into saline from concentrated stock just prior to injection.
5. Prepare complete culture medium for the cell line(s) being used.
   NOTE: For example, prepare medium for B16-F10 or A549 cells with DMEM 1x + 10% FBS + 1x glutamine substitute + 1x penicillin/streptomycin. Media must be kept sterile. Other cell line-specific media formulations can be used. Certain components such as glutathione may, in some cases, interfere with the onset of senescence. Empirical testing of various media formulations should be conducted if the onset of senescence is lower than expected or not observed with control chemotherapy agents.
6. Prepare staining and wash buffers.
   1. Prepare 1% bovine serum albumin (BSA) in 1x PBS for use in staining procedures. Dissolve 2 g of BSA into 200 mL of PBS and stir for 10 min, or until fully dissolved, at room temperature.
   2. Prepare 0.5% BSA in 1x PBS as a wash buffer. Dilute 100 mL of the 1% BSA prepared in step 1.6.1 into 100 mL of 1x PBS for 0.5% BSA.
   3. Store buffers at 4 °C for up to 1 month.
7. Prepare 4% paraformaldehyde in 1x PBS. Use commercially available, sealed paraformaldehyde ampules (e.g., 16% v/v) for convenience and stability: 2.5 mL of 16% PFA + 7.5 mL of 1x PBS (= 10 mL of 4% PFA). Adjust the prepared volume depending on the total volume needed per experiment.
   NOTE: Prepare as needed for cell fixation only; prepare fresh each time.
8. Prepare FACS sorting buffer: 1x PBS, 1 mM EDTA, 25 mM HEPES, 1% BSA (pH 7.2). Sterile-filter through a 0.22 µm filtration device and store at 4 °C for up to 1 month.
   NOTE: Prepare as needed for FACS sorting only. Flow-sorting buffer formulations may vary across FACS instruments. The above formulation is compatible with the instrument used in this study (see Table of Materials). Consult the manufacturer's guidelines.
9. Prepare tumor dissociation solution: 20 µg/mL Liberase TL + 100 µg/mL DNAse I in RPMI-1640 media (without FBS or other supplements). Stock solutions are useful to keep on hand of Liberase TL (prepared and stored as directed by the manufacturer) and DNAse I (100 mg/mL in double-distilled water [ddH₂O], stored at −20 °C).
   ​NOTE: Prepare as needed if using tumors only; prepare fresh each time.

2. Induction of senescence by chemotherapy drugs in cultured cancer cells

NOTE: All cell manipulation steps in this section should be performed in a biosafety cabinet using sterile practices. This section is written for adherent cell types. Suspension cells may be used with appropriate modifications as noted.

1. Grow cancer cell line(s) according to the standard protocol from the supplier or the laboratory that provided the specific cell line(s) used.
   NOTE: Low passage cells (p < 10) are generally preferred as there will be lower levels of replicative senescence, i.e. lower background, in untreated cell samples.
2. One day prior to senescence induction by drugs, harvest cells with trypsin-EDTA 0.25% (or as recommended). Neutralize trypsin by adding an equal volume of complete culture medium, and transfer the cell suspension to a sterile conical tube.
   NOTE: This step is not needed for suspension cells.
3. Count the cells using the standard hemacytometer method and record cells/mL. Plate cells at 1 × 10³-10 × 10³ cells/cm² in standard 6-well plastic culture dishes.
   NOTE: Optimal plating density is dependent on the proliferation rate of cells and should be user-determined. Cells should be in log phase growth at approximately 10%-20% confluency at the time of treatment (i.e., following 18-24 h incubation after plating). The starting density (cells/mL) of suspension cells should be user-determined. Six-well plates typically yield enough senescent cells per well for one standard flow cytometry analysis sample. If flow-sorting, a much larger surface area (e.g., multiple P150 plates) should be used to enable the recovery of sufficient numbers of senescent cells for downstream assays (≥1 × 10⁶).
4. Incubate plated cells overnight (18-24 h) in a 37 °C incubator with 5% CO₂ and a humidity pan.
5. Treat the plated cells with senescence-inducing chemotherapy drug(s). Include at least one positive control, e.g., etoposide (ETO) or bleomycin (BLM). Prepare duplicate wells per drug. Include one set treated with vehicle-only as control.
   NOTE: A dose curve for each experimental agent should be tested by the user to determine the optimal concentration for senescence induction in the cell line(s) being used.
6. Incubate cells for 4 days in a 37 °C incubator with 5% CO₂ and a humidity pan to allow the onset of senescence. Examine daily for expected morphology changes using a light microscope.
   NOTE: Incubation times from 3-5 days may be acceptable depending on the rate of senescence onset. Media can be changed and the agent reapplied (or not), as desired, to promote healthy growth conditions while achieving an acceptable percentage of senescent cells.
7. After the onset of senescence, harvest the cells by adding trypsin-EDTA 0.25% for 5 min at 37 °C. When cells are dissociated into suspension, neutralize trypsin with an equal volume of complete medium.
   NOTE: This step is not needed for cells growing in suspension. If surface marker staining will be conducted, avoid the use of trypsin-EDTA as it can temporarily destroy surface antigens on cells. Instead, gently dissociate the monolayer using a sterile plastic cell scraper (or an alternate dissociation reagent designed to preserve surface antigens).
8. Count the cells in each sample using a hemacytometer. Calculate the cells/mL for each sample.
   NOTE: Trypan blue can be added to evaluate the percentage of dead cells at this point (i.e., due to drug treatment), but cell death will also be determined with a fluorescent viability dye during DDAOG staining workflow.
9. Aliquot ≥0.5 × 10⁶ cells per sample into 1.7 mL microcentrifuge tubes.
   NOTE: The number of cells per sample should be standardized across all samples.
10. Centrifuge the tubes for 5 min at 1,000 × g in a microcentrifuge at 4 °C. Remove the supernatant.
    NOTE: If a refrigerated microcentrifuge is unavailable, it may be acceptable to perform centrifugations at ambient temperature for certain resilient cell types.
11. Proceed to DDAOG staining in section 4.

3. Induction of senescence by chemotherapy drugs in tumors established in mice

NOTE: If tumor cells will be FACS-sorted, ensure sterility at each step by working in a biosafety cabinet and working with sterile instruments, procedures, and reagents.

1. Create mouse tumor models by injecting cancer cells subcutaneously, according to standard methods (e.g., Appelbe et al.¹⁹).
   NOTE: The number of cancer cells to be injected, the injection site, and the appropriate mouse strain should be optimized for each protocol. Here, B16-F10 cells were injected subcutaneously at 1 × 10⁶ cells in 0.1 mL of saline (1 × 10⁷ cells/mL).
   1. Verify that the viability of cells using trypan blue is >90% before performing injections. Anesthetize the mice with isoflurane.
   2. Anesthetize 6-7-week-old female C57/BL6 mice with a mix of 3% isoflurane and air and maintain under these conditions in an induction chamber placed within a sterile biosafety cabinet. Confirm anesthesia by gently pinching the mouse's foot. Apply sterile veterinary ointment to both eyes to prevent corneal drying during the procedure. During the procedure, maintain mouse body temperature using a heating lamp.
   3. Working inside a sterile biosafety cabinet, remove the mouse from the induction chamber and place it in contact with a nose cone providing a 3% isoflurane supply. Shave the flank area at the injection site using a clean electric razor. Mix the prepared cell suspension briefly by manually inverting the tube just prior to injection, and inject the cell suspension subcutaneously into the shaved flank(s) using a sterile 0.5 mL syringe fitted with a sterile 27 G needle. Remove the mouse from the hood and transfer it to the recovery cage.
   4. In the recovery cage, monitor the vital signs of the mice continuously until they have regained sufficient consciousness to maintain sternal recumbency, demonstrate the righting reflex, and are able to safely move around in the cage. Do not leave mice unattended or return animals that have undergone tumor cell inoculation to the company of other animals until fully recovered. Monitor all inoculated mice daily for weight loss, reduced activity/mobility, and neurological symptoms; euthanize mice exhibiting severe symptoms in any category. For mice exhibiting pain symptoms following inoculation, administer buprenorphine (0.1-0.2 mg/kg) once subcutaneously.
      NOTE: Mice exhibiting persistent pain after buprenorphine should be euthanized.
2. Starting 5-7 days post cancer cell inoculation, measure tumor growth with calipers every 2-3 days. Initiate prosenescent treatment when the tumor has reached 50 mm³ ± 10 mm³ in volume.
   NOTE: In this work, doses of USP grade doxorubicin hydrochloride (DOX) or PEGylated liposomal doxorubicin (PLD) at 10 mg/kg were administered in 0.9% sodium chloride injection (USP). Drugs were injected intraperitoneally 3x, once every 5 days, starting when tumors reached 50 mm³ ± 10 mm³. Mice recovered for 7 days following the final treatment to allow the onset of TIS in tumors. Senescence induction dosages and conditions for other treatments and/or tumor models should be optimized.
3. At 7 days after the final drug treatment, sacrifice the mice by CO₂ overdose and cervical dislocation or other approved methods in compliance with laboratory animal work guidelines. Excise the tumors and collect them in sterile tubes or 6-well plates filled with sterile RPMI growth medium (to preserve viability during processing).
   NOTE: If performing a histological examination (e.g., X-Gal or immunohistochemistry), tumors can be bisected here, with one half snap-frozen in O.C.T. embedding medium and cryosectioned using standard procedures for frozen tissue histology. The remaining tumor half should yield abundant material for dissociation and DDAOG staining.
4. Transfer one tumor to a P100 plastic dish containing 5 mL of RPMI medium. Mince the tumor into pieces using a scalpel.
5. Transfer 5 mL of the suspension of tumor pieces containing suspended cells and debris into a 15 mL conical tube. Use the wider tip of a 25 mL serological pipet for transferring this suspension if large debris is present. Rinse the dish with an additional volume of sterile RPMI to collect materials. Cap and place the conical tube on ice.
6. Repeat steps 3.45-3.5 for the remaining tumors. Use a separate plate and scalpel for each tumor to avoid cross-contamination, or rinse well with PBS in between tumors. Use 5 mL of fresh medium for mincing each tumor.
7. Prepare the tumor dissociation solution: 20 µg/mL Liberase TL + 100 µg/mL DNAse I in RPMI-1640 media (without FBS).
   NOTE: Many effective formulations for tumor dissociation solutions exist and can include a variety of enzymes and other components from different manufacturers. Optimal concentrations of components can vary greatly across tumor types. If red blood cells are highly present in the tumor, red blood cell lysis may be additionally conducted; if dead cells are an issue, a dead cell removal kit may be used. It is highly recommended for the user to independently determine optimal tumor dissociation conditions that provide high viability and low presence of contaminating cells, connective material, and debris.
8. Centrifuge all tumor samples in conical tubes for 5 min at 1,000 × g (4 °C). Remove the supernatant.
9. Add 1-5 mL of tumor dissociation solution to each tumor sample, depending on the volume of tumor material. Ensure there is 1-2 mL in excess of the tumor material pellet in the tube. Vortex at a moderate speed to mix.
10. Place the samples in a 37 °C incubator with rapid rotation for 45 min. Vortex briefly every 15 min.
11. Filter each sample through a 100 µm cell strainer into a 50 mL conical tube. If the samples are too viscous to pass through the filter, add 10 mL of RPMI-1640 medium to dilute. Rinse the filters with RPMI medium to collect the residual cells.
12. Use a hemacytometer to count the cells/mL for each sample.
13. Aliquot two or more replicates of 5 × 10⁶ cells per tumor sample.
14. Centrifuge for 5 min at 1,000 × g (4 °C). Remove the supernatant.
15. (Optional) Cryopreserve the tumor samples for later DDAOG staining if desired.
    1. Resuspend the dissociated tumor cell pellet in cryopreservation medium: 50% FBS, 40% RPMI-1640, 10% DMSO, prepared under sterile conditions at 5 × 10⁶ cells/mL.
    2. Aliquot 1 mL of cell suspension into each cryovial.
    3. Freeze the cryovials for 24 h in an isopropanol cell freezing container at −80 °C; then, transfer to liquid nitrogen cryostorage for longer-term storage (>1 week).
    4. When staining is desired, thaw the cryovials on ice and proceed to DDAOG staining in section 4.
       NOTE: Some tumors may not remain viable through cryopreservation, and resilience to this process should be evaluated by the user for the tumor model of interest.
16. Proceed to section 4 for DDAOG staining.

4. DDAOG staining of SA-β-Gal in cell or tumor samples

1. Dilute 1 mM Bafilomycin A1 stock at 1:1,000x into DMEM medium (without FBS) for a final concentration of 1 µM.
2. Add prepared Baf-DMEM solution to the cell pellet samples (from step 2.11 or step 3.16) for a concentration of 1 × 10⁶ cells/mL.
   NOTE: For example, if using 0.5 × 10⁶ cells per sample, add 0.5 mL of Baf-DMEM. For tumors, 5 × 10⁶ cells can be stained in 5 mL of Baf-DMEM.
3. Incubate for 30 min at 37 °C (without CO₂) on a rotator/shaker set at a slow speed.
   NOTE: Avoid CO₂ incubators for the staining process, which can acidify solutions and thereby interfere with Baf and DDAOG staining.
4. Without washing, add the DDAOG stock solution (5 mg/mL) at 1:500x (10 µg/mL final) to each sample. Pipette to mix. Replace on a rotator/shaker at 37 °C (without CO₂) for 60 min. Protect from direct light.
5. Centrifuge the tubes for 5 min at 1,000 x g at 4 °C. Remove the supernatant.
6. Wash with 1 mL of ice-cold 0.5% BSA per tube and pipette to mix. Centrifuge the tubes for 5 min at 1,000 x g at 4 °C and remove the supernatant. Repeat this step 2x to thoroughly wash the cells. Remove the supernatant and proceed.
   NOTE: It is important to perform the wash steps in step 4.6 to remove uncleaved DDAOG, which can exhibit undesired fluorescence emission (460/610 nm).
   NOTE: If immunostaining for cell surface markers, proceed to section 5 below.
7. (Optional) Fixation and storage of DDAOG stained cells for later analysis
   1. Add 0.5 mL of ice-cold 4% paraformaldehyde dropwise to each washed sample. Pipette to mix.
   2. Incubate for 10 min at room temperature.
   3. Wash the cells 2x with 1 mL of PBS.
   4. Store the samples for up to 1 week at 4 °C prior to flow cytometry analysis.
      NOTE: For fixed samples, skip step 4.8.
8. Dilute Calcein Violet 450 AM stock (1 mM) at 1:1,000x into 1% BSA-PBS (1 µM final). Add 300 µL (for cultured cell samples) or 1,000 µL (for tumor samples) to the washed cell pellets from step 4.6. Incubate for 15 min on ice in the dark.
9. Proceed to flow cytometry setup (section 6).

5. (Optional) Immunostaining for cell surface markers in combination with DDAOG

NOTE: As with any flow cytometry experiment, single-stained control samples with DDAOG only and fluorescent antibody only should be prepared to determine crosstalk (if any) across fluorescence channels. If crosstalk is observed, standard flow cytometry compensation should be performed²⁰.

1. Resuspend the cell pellets obtained in step 4.6 in 100 µL of staining buffer (1% BSA in 1x PBS).
2. Add the Fc receptor blocking reagent appropriate for the cell species (mouse or human) at the manufacturer-recommended titration. Incubate for 10 min at 24 °C.
3. Add fluorophore-conjugated antibodies at the titration recommended by the manufacturer (or determined by the user). Incubate for 20 min on ice, protected from light.
4. Centrifuge the tubes for 5 min at 1,000 × g at 4 °C. Remove the supernatant.
5. Wash with 1 mL of ice-cold wash buffer (0.5% BSA-PBS) per tube and pipette to mix. Centrifuge the tubes for 5 min at 1,000 × g at 4 °C and remove the supernatant. Repeat this step 2x to thoroughly wash the cells.
6. Dilute 1 mM Calcein Violet 450 AM at 1:1,000x into 1% BSA-PBS. Add 300 µL to the washed cell pellets from step 5.5. Incubate for 15 min on ice in the dark.
7. Proceed to flow cytometry analysis (sections 6-7).

6. Flow cytometer setup and data acquisition

1. Transfer the cell samples to tubes compatible with the flow cytometry instrument. Place the tubes on ice and keep them protected from light.
   NOTE: If aggregates are observed in the cell suspensions, pass the suspension through 70-100 µm cell strainers prior to analysis. Do not use 40 µm strainers because they can exclude some of the larger senescent cells.
2. In the referenced software (see the Table of Materials), open the following plots: 1) FSC-A vs SSC-A dot plot, 2) violet channel histogram, 3) far-red channel (e.g., APC-A) versus green channel (e.g., FITC-A) dot plot.
   NOTE: Doublet exclusion plots and single-channel histograms can also be used but are not strictly required.
3. Initiate cytometer data acquisition.
   1. Place the vehicle-only control sample stained with DDAOG on the intake port. At a low intake speed, begin to acquire sample data.
   2. Adjust FSC and SSC voltages so that >90% of events are contained within the plot. If cells do not fit well on the plot, lower the area scaling setting to 0.33-0.5 units.
   3. Remove the vehicle-only sample without recording data.
   4. (Optional) Add one droplet of rainbow calibration microspheres to a cytometer tube with 1 mL of PBS. Place the tube on the cytometer intake port. Begin to acquire sample data.
   5. Adjust violet, green, and far-red channel voltages so that the top peak of the rainbow microsphere is in the range of 10⁴-10⁵ units of relative fluorescence in each channel and all peaks are well separated in each channel. Record 10,000 events. Remove the tube.
4. Place the positive control sample (e.g., BLM, ETO) stained with DDAOG on the intake port. At a low speed, acquire sample data. Observe the events in FSC, SSC, violet, green, and far-red channels to ensure that over 90% of events are contained within all plots. Look for an increase in AF and DDAOG signal versus vehicle-only control.
5. If using a sorting cytometer, initiate sorting at this step.
   1. For record-keeping purposes, record 10,000 cells for the control sample and each sorted sample.
   2. Sort the desired amount of cells (≥1 × 10⁶ is typically suitable) into an instrument-appropriate collection tube with 3-5 mL of culture medium.
   3. After sorting, proceed to downstream culture or analysis.
   4. Skip to section 7 for the routine analysis of sorted samples.
6. If using fluorescent antibodies, optimize channel voltages here using the unstained, single-stained, and double-stained samples prepared in section 5.
   NOTE: For the calibrated flow cytometer used herein, optimal channel voltages typically fell between 250 and 600 (mid-range), but the optimal voltages and channel voltage ranges will vary across instruments. Avoid using voltages at very low or high ranges, which may suppress signal or amplify noise.
7. After completing steps 6.1-6.5 and making adjustments to the cytometer settings as necessary, record data for all the samples. Ensure that the settings remain uniform for all sample recordings. Record ≥10,000 events per cultured cell sample or ≥100,000 events per tumor cell sample.
   NOTE: Although gating and analysis can be performed using data acquisition software (e.g., FACSDiva), a complete gating and analysis workflow to be conducted post acquisition using separate analysis software (FlowJo) is described in section 7 below. Post-acquisition analysis is preferred to reduce time at the cytometer workstation and take advantage of additional tools included in the dedicated analysis software.
8. Save sample data in .fcs file format. Export the files to a workstation computer equipped with flow cytometry analysis software (e.g., FlowJo). Proceed to section 7.

7. Flow cytometry data analysis

NOTE: The workflow presented uses FlowJo software. Alternative flow cytometry data analysis software may be used if the key steps described in this section are similarly followed.

1. Using FlowJo software, open .fcs data files from step 6.7.
2. Open the layout window.
3. Drag and drop all samples into the layout window.
4. Gate viable cells.
   1. First double-click on the sample data for the vehicle-only control to open its data window.
   2. Visualize the data as a violet channel histogram. Identify the viable cells stained by CV450 based on their brighter fluorescence than the dead cells.
   3. Draw a gate using the single-gate histogram tool to include viable cells only. Name the gate viable.
   4. Then, from the sample layout window, drag the viable gate onto the other cell samples to apply the gate uniformly.
   5. In the layout window, visualize all samples as violet channel (viability) histograms. Verify that viable cell gating is appropriate across samples before proceeding; if not, make adjustments as needed.
      NOTE: Viability staining can exhibit variations across treatments or tumors.
5. Gate senescent cells.
   1. Double-click on the gated viable cell data for the vehicle-only control to open its data window.
   2. Visualize the data as a dot plot for far-red channel (DDAOG) vs green channel (AF).
   3. Draw a gate using the rectangle gating tool to include <5% of cells that are DDAOG⁺ and AF⁺ (upper right quadrant). Name the gate senescent.
   4. Then, from the sample layout window, drag the senescent gate onto the viable subsets of the other cell samples to apply the gate uniformly.
   5. Into the layout window, drag and drop all viable cell subsets gated in section 7.4. Visualize all viable samples as far-red (e.g., APC-A) versus green channel (e.g., FITC-A) dot plots.
   6. Ensure that the senescent gate drawn in step 7.5.3 is visible on all plots and that the gate for the vehicle-only control exhibits ≤5%-10% senescent cells.
6. Once the percentage of senescent cells has been determined using the steps above, present the resulting data using the FlowJo plots, summarized in a data table and/or statistically analyzed using standard software.

Representative Results

Several experiments were performed to demonstrate the comparability of DDAOG to X-Gal and C₁₂-FDG for the detection of senescence by SA-β-Gal. First, X-Gal was used to stain senescent B16-F10 melanoma cells induced by ETO (Figure 2A). An intense blue color developed in a subset of ETO-treated cells, while other cells exhibited less intense blue staining. Morphology was enlarged in most ETO-treated cells. Staining ETO-treated cells with fluorescent SA-β-Gal substrate C₁₂-FDG (green) or DDAOG (far-red) demonstrated comparable staining patterns and intensity variations to X-Gal (Figure 2B). However, green C₁₂-FDG emission overlapped with cellular AF (Figure 2C), which is known to accumulate in senescent cells¹⁷. In contrast, AF was negligible in the far-red emission range of DDAOG.

Instead of using a fluorescence microscope to score and count senescent cells, it was more expedient to take advantage of the high-throughput capabilities of a flow cytometer to acquire data for thousands of cells per sample in a short time (<5 min per sample). First, a series of standard flow cytometry setup parameters were implemented to ensure optimal data acquisition (Figure 3). Following a typical approach, light scatter parameters were set to visualize the volume (forward scatter, FSC) and granularity (side scatter, SSC) of cells (Figure 3A). Here, we noted the trend of increased volume of ETO-treated cells, which agreed with the enlarged morphology typically observed for senescent cells using microscopy. A reduction in default area scaling settings (to 0.33-0.50 units depending on the cell type and treatment) was necessary to visualize more of the larger cells on the plot. For some cell lines/treatments, increased granularity (SSC) was also evident (data not shown). Overall, scatter evaluation was used as a quality control step to verify that cell scatter data appeared as expected, excessive cell debris was not present, and cells were being processed through the cytometer at appropriate flow rates (~100-1000 cells/s). As a quality control step used only for instrument setup, no gating or analysis was performed here.

A second (optional) step in the flow cytometry setup was to briefly analyze a sample of commercially available "rainbow" calibration particles to ensure the fluorescent detection voltages were set in acceptable ranges (Figure 3B). The brightest peak was set between 1 × 10⁴ units and 1 × 10⁵ units of relative fluorescent intensity in each channel, with clearly defined lower intensity peaks, sufficient separation between each peak, and no overlap of neighboring peaks. A control sample of 10,000 microspheres was recorded using these voltage settings. The microspheres were then used in this manner in each cytometry session to improve the uniformity of data acquisition over the course of the protocol.

Next, samples of vehicle-only or ETO-treated cells were visualized in each fluorescent channel, and gates were set. Cell viability gates were set based on the signal of CV450 viability stain in the violet channel (Figure 3C). Vehicle-only treated cells exhibited 88% viability, and ETO-treated cells exhibited 75% viability (in the final cell sample; additional dead cells were likely initially removed in discarded culture media and mechanically disintegrated during the staining process). Next, viable gated cells were visualized in the green (Figure 3D) and far-red (Figure 3E) emission channels. Gates for green AF^HI and far-red DDAOG^HI were set at <5% of vehicle-only cells, and these gates were then applied to ETO-treated cells. Using this approach, it was determined that 46% of ETO-treated cells were AF^HI and 33% were DDAOG^HI; these values were in the expected range based on literature and from results of numerous replicate experiments in our laboratory. Once the cytometer setup was complete, all cell samples in the experiment were run using identical data acquisition settings. Data for 10,000 events per sample were obtained.

Representative assay data is shown in Figure 4. B16-F10 murine melanoma cells or A549 human lung adenocarcinoma cells were used as the cancer cell line models. Each cell line was treated with a chemotherapy agent known to induce senescence (ETO or BLM) for 4 days to induce senescence or vehicle-only. Further, the known senolytic agent ABT-263²¹ was added to induced senescent cells for 2 days to demonstrate the specificity of the DDAOG probe. ABT-263 only samples were prepared as additional controls. Here, data are visualized as 2D dot plots with DDAOG (670 nm emission) versus AF (525 nm). The workflow from Figure 3 was used for cytometer setup, and a TIS gate was set such that <5% of vehicle-only cells scored as TIS. Results using B16-F10 cells (Figure 4A) showed that ETO induced TIS in 35% of viable B16-F10 cells (similar to the comparable data shown in Figure 2D,E) and the senolytic agent almost completely eliminated TIS cells (<2%). In A549 cells (Figure 4B), BLM induced TIS in 66% of viable cells, and ABT-263 reduced the percentage to 15%. ABT-263 alone was not toxic to untreated, proliferating cells.

We further aimed to demonstrate that fluorescent antibody co-staining was compatible with the DDAOG senescence assay to facilitate the screening of TIS-associated or novel surface markers. Here, B16-F10 mouse melanoma cells were again treated with TIS-inducing ETO (or vehicle) for 4 days. Then, cells were both stained with DDAOG to evaluate TIS and with a fluorescent antibody to detect the TIS-associated surface marker DPP4²² (Figure 5). Anti-DPP4 conjugated to R-phycoerythrin (PE) was used, and we confirmed negligible overlap of PE with DDAOG and AF on the flow cytometer used (Supplementary Figure S2). A histogram of PE channel data (Figure 5A) for >5,000 viable cells showed that 42% of ETO-treated cells were DPP4⁺ (using the vehicle-only sample to set the positivity gate). Visualizing 2D dot plots for the same samples (Figure 5B,C) indicated that 44% of ETO-treated cells were double-positive for DDAOG (i.e., senescent) and DPP4 versus 4% of vehicle-only cells. These data demonstrate that live-cell staining with surface marker antibodies is possible in combination with the DDAOG senescence assay.

Potential concerns with any live cell staining method include cell death occurring during lengthy analysis sessions (>1 h) and how to most efficiently stain and analyze samples across many different time points (up to days apart). Solvent-based fixation of stained cells addresses both of these concerns, as samples can be fixed as soon as they are stained and then stored in the refrigerator until analysis in one temperature-stable batch. Thus, we tested whether live cells stained with DDAOG could be fixed with 100% methanol or 4% paraformaldehyde (PFA) and stored for up to 1 week at 4 °C. Undesirably, methanol fixation decreased the DDAOG signal and significantly reduced AF (data not shown); hence, the use of methanol as a fixation solvent should be avoided. However, fixation with PFA was much more successful, as seen in Figure 6 for cells fixed in 4% PFA for 10 min after staining with DDAOG. Compared to unfixed control samples (Figure 6A; untreated, 5% and BLM, 67% DDAOG^HI AF^HI), fixed samples (Figure 6B) exhibited slightly higher background in untreated cells (9%) and also a higher percentage of cells scoring as senescent in BLM-treated cells (80%). This effect was also seen in fixed samples stored overnight (Figure 6C; untreated, 12% and BLM, 72%) and stored for 1 week at 4 °C (Figure 6D; 14% and 70%). Despite the slight increases in fluorescence caused by PFA fixation, the induction of senescence by BLM was still evident in all fixed samples versus the matched untreated sample. Further, cells remained intact in storage with only slight deterioration by Day 7, and no problematic aggregation was observed. We conclude that the convenience of being able to fix and store cell samples for later batched analysis will justify tolerating the slightly higher background caused by PFA fixation, particularly in experiments with many samples or time points.

A common challenge in cell-based senescence research is the heterogeneous onset of senescence in cell populations. Here, we show that DDAOG can be used for FACS of viable senescent cells and that collected cells survive in culture for downstream in vitro assays (Figure 7). To sort cells by FACS, cells are treated and stained, as described, and sorted by a FACS-capable flow cytometer using the DDAOG versus AF gating strategy shown here (Figure 7A). As a viability probe is not used here due to long-term toxicity concerns, we recommend stringent scatter gating to be performed prior to the final senescent cell gating (Supplementary Figure S3) to eliminate false-positive debris from the collected cells. These are standard FACS gating procedures that should be familiar to a moderately experienced user and can be rapidly established using software at the instrument (<10 min).

After sorting a sample of BLM-treated A549 cells, we returned the cells to culture in standard multiwell dishes for 5 days (n = 6 replicates) at 10 × 10³ cells/cm². Unsorted controls were plated at the same density, grown alongside, and untreated and BLM-treated cells were included. Cells were observed daily; for sorted cells, no significant cell death or return to proliferation was observed. Sorted cells remained sparse (i.e., not proliferating) over the course of 5 days in culture, while untreated and BLM-treated cells became confluent as shown. On Day 5, cells were fixed in PFA and stained for morphology and proliferation markers (Figure 7B). The characteristic enlarged morphology of sorted cells was revealed by fluorescent Phalloidin staining of filamentous actin, with DAPI staining to show enlarged nuclei. Sorted cells were very large in diameter (>10 µm) with a characteristic rounded appearance. As expected, Ki67 antibody staining revealed a complete loss of the proliferation marker in the sorted sample versus a partial loss in the unsorted BLM-treated sample, and normal levels of Ki67 were seen in many cells in the untreated-unsorted sample. At least three images were taken per well using uniform imaging settings across samples. Representative images are shown (Figure 7B).

Finally, we assessed whether senescent cells arising in tumors established in mice treated with chemotherapeutic drugs could be identified using DDAOG. B16-F10 melanoma tumors were induced in the flank of C57/BL6 mice, which were then treated three times (i.p., every 5 days) with saline only (Figure 8A), DOX (Figure 8B), or PLD (Figure 8C). After the third treatment, the mice recovered for 7 days to allow the onset of senescence and were then sacrificed and tumors excised. Tumors were halved, with half used to prepare frozen tissue slides for X-Gal staining (Figure 8) and half dissociated into single-cell suspensions and stained with DDAOG (Figure 8 and Supplementary Figure S4). X-Gal staining in tissues was rather weak despite a long staining duration (72 h), but blue staining was evident in DOX and PLD tumors upon close inspection, particularly in tumors that also scored positive for senescence by DDAOG flow assay (Figure 8B,C). At least three images were taken per tissue slide and representative images are shown.

Compared to X-Gal, DDAOG was a more sensitive and accurate way to quantify senescence in tumors (Figure 8). For DDAOG tumor analysis, the saline-treated tumor with the highest fluorescence background was used to set the senescence gate at <5%, such that the other saline-treated tumors did not score above 5% senescence. This senescence gate was then batch-applied to gated viable cells from all tumors. Both chemotherapy agents induced senescence in some tumors (two of five tumors for DOX, three of five for PLD), with percentages of senescent tumor cells varying from 3% to 36% per tumor. Cytometry data plots for all 15 tumors are shown in Supplementary Figure S4, and a summary of tumor cytometry data is shown in Figure 8D (n = 5; (*) p < 0.05 vs. saline-only control group by the F test of variance). We conclude that DDAOG versus AF flow cytometry is an acceptable method for screening tumors and chemotherapy agents for the induction of senescence in vivo.

Figure 2: DDAOG is a sensitive and specific stain for SA-β-Gal. (A) Conventional staining for SA-β-Gal using X-Gal showing untreated (left) or ETO-treated (right) B16-F10 murine melanoma cells. Senescence induced by ETO: cells exhibit enlarged morphology and blue staining due to cleavage of X-Gal by elevated SA-β-Gal. Scale bars = 10 µm. (B) Fluorescent staining for SA-β-Gal in ETO-treated cells using either C₁₂-FDG (515 nm emission, green) or DDAOG (660 nm, red). Staining distribution is similar for both probes, indicating that DDAOG detects SA-β-Gal in a similar manner to C₁₂-FDG. (C) Evaluation of AF in unstained, ETO-treated cells in either the green (525 nm emission, left) or far-red (660 nm, right) emission channel. AF from lipofuscin is high in the green emission channel and negligible in the far-red channel. Exposure time = 2,000 ms for each image. Scale bars = 10 µm. This figure is reprinted from Flor and Kron²³. Abbreviations: DDAO = 9H-(1,3-dichloro-9,9-dimethylacridin-2-one); DDAOG = DDAO-Galactoside; X-Gal = 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside; UNT = untreated; ETO = etoposide; AF = autofluorescence; C₁₂-FDG = 5-dodecanoylaminofluorescein di-β-D-galactopyranoside; SA-β-Gal = senescence-associated beta-galactosidase. Please click here to view a larger version of this figure.

Figure 3: Flow cytometer data acquisition setup. (A) Representative scatter plot distribution of cells. FSC-A is a readout of cell volume, and SSC-A indicates cellular granularity. Left panel, vehicle-only treatment of A549 cells. Right panel, ETO treatment to induce senescence. Note the trend toward enlarged cell volume of ETO-treated cells, in agreement with the enlarged morphology evident from microscopy. (B) Optional use of 5-peak commercial "rainbow" fluorescent calibration microspheres to set detector voltages of the cytometer. In each fluorescence channel used, the maximum peak should be set ≤1 x 10⁵ relative fluorescent units by adjusting the cytometer detector voltage while samples are running. Left panel, BV421 (violet) channel; center, FITC (green) channel; right, APC (red) channel. The five fluorescent peaks should exhibit distinct spacing as shown. (C-E) Representative single-channel fluorescence data for (C) viability staining using violet CV450 dye; (D) green autofluorescence of cells; (E) far-red signal from DDAOG, detecting SA-β-Gal. Darker color histogram, vehicle-only treatment; lighter color histogram, ETO treatment. Parent gates are indicated above each plot. Abbreviations: FSC-A = forward scatter-peak area; SSC-A = side scatter-peak area; ETO = etoposide; BV421-A = Brilliant Violet 421 channel peak area; FITC-A = fluorescein isothiocyanate channel peak area; APC-A = allophycocyanin channel peak area; AF = autofluorescence; DDAO = 9H-(1,3-dichloro-9,9-dimethylacridin-2-one); DDAOG = DDAO-Galactoside; VEH = vehicle. Please click here to view a larger version of this figure.

Figure 4: Representative data for flow cytometry senescence assay. (A) B16-F10 murine melanoma cells treated with (upper left) vehicle only, (upper right) ETO, (lower left) ABT-263 (1 µM) only, or (lower right) ETO plus ABT-263. (B) A549 human lung adenocarcinoma cells treated with (upper left) vehicle only, (upper right) BLM, (lower left) ABT-263 only, or (lower right) BLM plus ABT-263. (A,B) Rectangular gates in upper right quadrants of all plots define senescent cells (DDAOG^HI AF^HI). The percentage of senescent cells (of total viable cells per sample) is indicated on each plot. Abbreviations: DDAO = 9H-(1,3-dichloro-9,9-dimethylacridin-2-one); DDAOG = DDAO-Galactoside; ETO = etoposide; BLM = bleomycin; VEH = vehicle; TIS = therapy-induced senescence. Please click here to view a larger version of this figure.

Figure 5: Co-staining of an example cell surface marker with senescence assay. (A) Immunodetection of senescence marker DPP4 on the surface of viable B16-F10 cultured cells; dark orange, vehicle only; light orange, ETO. (B,C) Center and right panels: cells co-stained with DDAOG and anti-DPP4:PE. DDAOG^HI DPP4^HI cells are contained within rectangular gates shown on the plots, and the percentage of double-positive cells per sample is indicated. Shown: viable-gated cells. Abbreviations: DDAO = 9H-(1,3-dichloro-9,9-dimethylacridin-2-one); DDAOG = DDAO-Galactoside; ETO = etoposide; VEH = vehicle; DPP4 = dipeptidyl peptidase 4. Please click here to view a larger version of this figure.

Figure 6: Poststaining fixation and storage of DDAOG-stained cell samples for later analysis. (A) Control, unfixed samples of live A549 cells either untreated (left) or treated with BLM (right) to induce senescence, and then stained and immediately analyzed using the DDAOG protocol (without fixation, Day 0). (B) Samples prepared as in (A) and then immediately fixed in 4% paraformaldehyde and analyzed (Day 0). (C) Samples prepared as in (A), immediately fixed in 4% paraformaldehyde, and stored overnight at 4 °C prior to analysis. (D) Samples prepared as in (A), immediately fixed, and stored for 7 days prior to analysis. Rectangular gates in the upper right quadrants of all plots define senescent cells (DDAOG^HI AF^HI). The percentage of senescent cells is indicated on each plot. Abbreviations: TIS = therapy-induced senescence; DDAO = 9H-(1,3-dichloro-9,9-dimethylacridin-2-one); DDAOG = DDAO-Galactoside; BLM = bleomycin; AF = autofluorescence. Please click here to view a larger version of this figure.

Figure 7: Flow cytometric sorting and validation of enriched senescent cell populations. (A) Flow cytometric sorting data showing (left) the untreated, DDAOG-stained control used to set the gate for senescent cell sorting (<5% senescent cells in gated region) and (right) the BLM-treated, DDAOG-stained sample that was sorted using the sort gate as shown. (B) Fluorescence microscopy images for (left column) cells stained with Phalloidin-Alexa Fluor 647 (orange pseudocolor) to detect F-actin and DAPI (blue) to counterstain nuclei, demonstrating enlarged morphology of senescent cells, or (right column) cells stained with rabbit Ki67 antibody and anti-rabbit Alexa Fluor 594 to detect loss of proliferation in senescent cells. Top row, untreated and unsorted cells. Center row, BLM-treated and unsorted cells. Lower row, BLM-treated and sorted cells. Scale bars = 10 µm. Abbreviations: TIS = therapy-induced senescence; DDAO = 9H-(1,3-dichloro-9,9-dimethylacridin-2-one); DDAOG = DDAO-Galactoside; BLM = bleomycin; UNT = untreated; DAPI = 4',6-diamidino-2-phenylindole. Please click here to view a larger version of this figure.

Figure 8: Quantification of senescence in tumors from mice treated with chemotherapy drugs. B16-F10 melanoma tumors were established in the flanks of C67BL/6 mice, which were then treated with three doses of (A) saline, (B) DOX, or (C) PLD every 5 days plus 1 week to allow the onset of senescence. Tumors were excised and halved; frozen tissue slides were prepared from one half for X-Gal staining (top row), and the other half was dissociated for DDAOG staining (lower row). In X-Gal-stained images, blue cells are SA-β-Gal ^HI senescent cells, while brown regions are due to melanin in tumors. Representative results are shown for DOX and PLD from two tumors per group that exhibited senescence. All saline-only tumors exhibited negligible senescence. (D) Quantification of senescence in drug-treated tumors from mice. (*) p < 0.05 by F test of variance, n = 5 mice per group. Error bars, mean ± SEM. Abbreviations: DDAO = 9H-(1,3-dichloro-9,9-dimethylacridin-2-one); DDAOG = DDAO-Galactoside; DOX = doxorubicin; PLD = PEGylated liposomal doxorubicin; X-Gal = 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside; AF = autofluorescence. Please click here to view a larger version of this figure.

Supplementary Figure S1: Chemical structure of the DDAOG probe. DDAOG is a conjugate of 7-hydroxy-9H-(1,3-dichloro-9,9-dimethylacridin-2-one) and beta galactoside. When cleaved by beta-galactosidase, the hydrolyzed cleavage product exhibits a 50 nm far-red fluorescence emssion shift, enabling its specific detection with excitation above 600 nm. Abbreviations: DDAO = 9H-(1,3-dichloro-9,9-dimethylacridin-2-one); DDAOG = DDAO-Galactoside. Please click here to download this File.

Supplementary Figure S2: Spectral scan of DDAOG crosstalk with other fluorescent channels of the flow cytometer. To identify available channels for the detection of fluorescent antibodies, a spectral scan was performed on a 4-laser, 15-channel flow cytometer using A549 cells treated with BLM and stained with DDAOG (red) or unstained (black). Data in every channel of the flow cytometer were acquired for 10,000 cells. (A) Emission channels of the 405 nm laser: from left to right, BV421, BV510, BV605, BV660, and BV711. The crosstalk observed in BV605, BV660, and BV711 channels makes them unsuitable for co-staining with DDAOG without compensation. BV421 and BV510 are suitable for co-staining (note that BV421 is typically used for viability staining). (B) Emission channels of the 488 nm laser: FITC and PerCP-Cy5. High crosstalk was observed for the PerCP-Cy5 channel. FITC is suitable for co-staining; however, note that the FITC channel is typically used in the DDAOG assay for the evaluation of green emission AF. (C) Emission channels of the 561 nm laser: PE, PE-Dazzle 594, PE-Cy5, PE-Cy5.5, and PE-Cy7. The PE and PE-Dazzle 594 channels are suitable for the detection of antibodies labeled with these fluorophores (PE is demonstrated for the detection of DPP4 in this study). (D) Emission channels of the 640 nm laser: APC, APC-H700, and APC-Cy7. The DDAOG signal of the senescent cells is visible in the APC channel (47.8% senescent). The signal overlaps into the APC-H700 and APC-Cy7 channels, making them unsuitable for co-staining without significant spectral compensation. Abbreviations: BLM = bleomycin; BV = Brilliant Violet; FITC = fluorescein isothiocyanate; PerCP = peridinin-chlorophyll protein; PE = phycoerythrin; DZ594 = Dazzle 594; APC = allophyocyanin; AF = autofluorescence; DDAO = 9H-(1,3-dichloro-9,9-dimethylacridin-2-one); DDAOG = DDAO-Galactoside. Please click here to download this File.

Supplementary Figure S3: Cell gating strategy for FACS sorting of senescent cells. Passing a suspension of cells through a sensitive FACS cytometer can require additional gating to ensure the purity of sorting and optimal instrument functionality. An example strategy is shown here. Other strategies are possible depending on manufacturer recommendations for the FACS cytometer being used. Left column, vehicle-only treated cell control. Right column, A549 cells treated with BLM to induce senescence to be sorted. (A) FSC-A (cell volume) versus SSC-A (cell granularity) gating of intact cells. The intact gate eliminates cell debris from the sorted sample. (B) FSC-A versus FSC-H purity gating; removes doublets and debris. (C) SSC-A versus SSC-H purity gating; removes doublets and debris. (D) Gating for the population of interest using the APC-A channel (637 nm excitation, 670 nm ± 30 nm emission, used for DDAOG) versus FITC-A channel (488 nm excitation, 530 nm ± 30 nm emission, used for AF); removes possible staining artifacts. (E) Senescent cell gating for final sorting. Abbreviations: FACS = fluorescence-activated cell sorting; BLM = bleomycin; FSC-A = forward scatter-peak area; SSC-A = side scatter-peak area; FSC-H = forward scatter-peak height; SSC-H = side scatter-peak height. Please click here to download this File.

Supplementary Figure S4: DDAOG senescence flow cytometry staining of tumors. Flow cytometry data for ≥50,000 viable tumor cells. Senescent cell gate, upper right quadrant of each plot. The percentage of senescent (of viable) tumor cells is shown within the gate. Tumor treatments included (A) saline-only; (B) DOX; and (C) PLD. Mice were treated three times, once every 5 days, with 7 days for recovery to allow the onset of senescence before sacrifice. Five tumors per condition were analyzed. Abbreviations: AF = autofluorescence; DDAO = 9H-(1,3-dichloro-9,9-dimethylacridin-2-one); DDAOG = DDAO-Galactoside; DOX = doxorubicin; PLD = PEGylated liposomal doxorubicin. Please click here to download this File.

Discussion

Over the last decade or so, flow cytometry has become a more common assay platform in cancer research due to the emerging popularity of tumor immunology, the development of lower-cost flow cytometers, and the improvement of shared instrumentation facilities at academic institutions. Multicolor assays are now standard, as most newer instruments are equipped with violet, blue-green, and red to far-red optical arrays. Thus, this DDAOG protocol is likely to be compatible with a wide array of flow cytometers. Of course, any flow cytometer should be user-evaluated. Particular care should be taken when adding additional fluorophores (e.g., fluorescent, conjugated antibodies) to the DDAOG assay. An evaluation of fluorophore crosstalk between channels should be conducted using single-stained controls visualized in all other relevant channels. If overlap is observed, spectral compensation can be performed²⁰ for correction following typical methods.

The findings shown herein are primarily intended to demonstrate that the DDAOG flow cytometry assay can produce rapid, quantitative, easy-to-interpret results for TIS induced by chemotherapy drugs in cells or tumors. The agents used here, including ETO^23,24, DOX^7,25, and BLM^26,27, have been documented to induce TIS in various cancer cell lines²⁴. To demonstrate the specificity of the DDAOG probe, the known senolytic agent ABT-263²¹ was demonstrated to selectively eliminate senescent cells in culture. This paper demonstrates the use of one murine (B16-F10) and one human (A549) cultured cancer cell line, as well as B16-F10 tumors established in mice. However, any cells that express β-Gal and retain viability through a standard flow cytometry sample preparation can be used. Certain cell types may be more fragile or less prone to TIS, and this should be evaluated before embarking upon a large screen or study. If cells disintegrate in preparation, viability is poor, or TIS is much lower than expected using positive agent controls, the cell type may not be an ideal model for studies of senescence. The agents and cell lines shown here can be used by other groups as controls in further screening of potential TIS-inducing agents or novel senolytics, which remains an active goal in the field.

Toxicity induced by chemotherapy drugs can vary across cell types and affect assay results. If the primary observed effect of the agent and/or concentration used is acute cell death, overall senescence may be minimal. In vivo, high tumor necrosis or systemic toxicity in animals is to be avoided by lowering the agent dosage. The key to assay success is the testing of a range of agent concentrations, with careful inspection of viability assay data (e.g., as provided by CV450 within the DDAOG assay). In vitro, if many dead cells detach from the plate during treatment, including the culture medium in the analyzed sample, it is important to evaluate cell death in total. CV450 is not the sole viability stain that is compatible with this assay; other violet/blue emitting fluorophores may be used. Fixable viability probes can also be used if the user plans to fix the stained samples with PFA, provided the probe fluorescence does not significantly overlap with DDAOG or AF (or the user conducts spectral compensation to correct for overlap). In some cases where agent toxicity is low and cells are robust, gating intact cells by light scatter (FSC vs. SSC) can be sufficient to isolate "viable" cells for analysis.

A key step in this in vitro protocol is to plate cells in the lower range of log growth density (typically 2 × 10³-5 × 10³ cells/cm²) to allow rapid initial proliferation, which facilitates the uptake of the chemotherapy drug by most cells. Once treated, it is also important to allow time for the onset of TIS: in vitro, 4 days ± 1 day in the presence of the drug; in vivo, 7 days of recovery following the final chemotherapy treatment. After staining with DDAOG as described, quantitative analysis of cytometric data should be performed as shown, i.e., gating DDAOG versus AF cells in each sample to determine the percentage of senescent cells (of total viable). Optional steps include the use of fluorescent "rainbow" calibration microspheres to standardize flow cytometry, PFA fixation of stained samples, co-immunostaining for surface markers, and flow sorting to enrich senescent cells for downstream assays. However, each of these optional steps provides key advantages in certain applications. The calibration microspheres standardize the cytometry setup across multiple sessions and allow the user to initially set voltages in a useful range for senescence and viability detection, with minimal adjustments thereafter. PFA fixation of samples stabilizes the cells and allows batched analysis of large sets of cells at a later time. Co-immunostaining for surface markers can be used to screen for novel senescence-related proteins and immune interactions. In future studies, we plan to validate the co-staining of intracellular senescence markers with DDAOG.

Sorting TIS cells by FACS allows for the enrichment of viable TIS cells from heterogeneous populations, which can confound readouts for downstream assays such as western blotting, proteomics, or transcriptomics. After sorting, no significant toxicity of DDAOG was observed in TIS cells placed back into culture for up to 5 days. However, it should be taken into consideration that sorted cells have been treated with Baf, internalized DDAOG (which is cleaved by SA-β-Gal to DDAO, an acridine dye), and been subjected to mechanical stress passing through the FACS instrument. Therefore, certain biological alterations not related to senescence may be present in the sorted cells. However, in this study, the sorted cells retained strong features of senescence and provided characteristic proteomics and transcriptomics results²⁸ when compared to unsorted controls. Despite the somewhat obvious utility of using FACS to collect and analyze senescent cells from tumors, this procedure has rarely been used in the literature. Some groups have used p16^Ink4a luciferase or fluorescent reporters to identify senescent cells in mouse tissues, with fluorescent reporters enabling FACS sorting in some studies^29,30. Findings generally agree that, regardless of the induction agent or tumor type, TIS in tumors is a partial to rare occurrence, rarely reaching 100% of tumor cells³¹. FACS sorting of cells using the DDAOG method readily allows the collection of rare TIS cells from tumors, without the need to express transgenic constructs.

Currently, most senescence research in mouse models is conducted ex vivo using a combination of X-Gal and immunohistochemistry markers^32,33. Yet, assessing TIS ex vivo using tumor tissues can be a lengthy process, particularly when X-Gal is used. This procedure requires tumor cryopreservation, cryosectioning onto slides, X-Gal staining, cover glass mounting, drying, imaging, and scoring "blue" cells-a multi-day approach at minimum. Immunohistochemistry is not much faster or easier, and different tissue sections must be used and scored for each marker plus X-Gal in each tumor, unless the multiplexed analysis is optimized, adding more time at the front-end of the process. Tissue sections sample only one thin cross-section of the tumor, while senescent cells (like many cell types) may be distributed unevenly in 3D space throughout tumors²⁹. It is urgently needed in the field to move away from slow, outdated histology methods toward a more rapid and readily quantifiable senescence assay for tumors. Using DDAOG flow cytometry, a set of 15 tumors could be dissociated and stained to obtain conclusive, quantitative senescence data in less than 1 day of hands-on time following tumor harvest. One half of the tumor was processed for each sample, improving the sampling of 3D space per tumor. This DDAOG flow cytometry approach is significantly faster and more reliable than tissue slide-based methods for the evaluation of TIS in tumors.

With its many advantages over X-Gal and other methods, we advocate for the DDAOG protocol to become the new gold standard senescence assay. It uses both the conventionally accepted senescence marker, SA-β-Gal, and label-free detection of age-associated AF as a second marker. These fluorophores are compatible with most standard flow cytometers. The assay includes a viability stain to exclude dead cells and debris using samples readily obtained from drug-treated cultured cell lines or tumors. Live cell samples can optionally be solvent-fixed or cryopreserved to facilitate the batched analysis of large studies, e.g., using time points to evaluate the onset of TIS over time using multiple agents The staining process takes less than a half-day of lab work to complete, and data acquisition is typically <5 min per sample. Data analysis is similarly rapid and straightforward, producing quantitative data on the percentage of TIS cells per sample without tedious cell scoring and counting. Samples can be FACS-sorted to recover enriched populations of TIS cells, which reduces noise from cellular heterogeneity in downstream analyses. We believe the DDAOG assay can, in many cases, replace X-Gal, facilitating the screening of TIS-inducing agents and senolytics in vitro and in vivo, leading to faster and more reliable discoveries in the field of senescence research.

Materials

Name	Company	Catalog Number	Comments
Bafilomycin A1	Research Products International	B40500
Bleomycin sulfate	Cayman	13877
Bovine serum albumin (BSA)	US Biological	A1380
Calcein Violet 450 AM viability dye	ThermoFisher Scientific	65-0854-39	eBioscience
DPP4 antibody, PE conjugate	Biolegend	137803	Clone H194-112
Cell line: A549 human lung adenocarcinoma	American Type Culture Collection	CCL-185
Cell line: B16-F10 mouse melanoma	American Type Culture Collection	CRL-6475
Cell scraper	Corning	3008
Cell strainers, 100 µm	Falcon	352360
DDAO-Galactoside	Life Technologies	D6488
DMEM medium 1x	Life Technologies	11960-069
DMSO	Sigma	D2438
DNAse I	Sigma	DN25
Doxorubicin, hydrochloride injection (USP)	Pfizer	NDC 0069-3032-20
Doxorubicin, PEGylated liposomal (USP)	Sun Pharmaceutical	NDC 47335-049-40
EDTA 0.5 M	Life Technologies	15575-038
Etoposide	Cayman	12092
FBS	Omega	FB-11
Fc receptor blocking reagent	Biolegend	101320	Anti-mouse CD16/32
Flow cytometer (cell analyzer)	Becton Dickinson (BD)	Various	LSRFortessa
Flow cytometer (cell sorter)	Becton Dickinson (BD)	Various	FACSAria
GlutaMax 100x	Life Technologies	35050061
HEPES 1 M	Lonza	BW17737
Liberase TL	Sigma	5401020001	Roche
Paraformaldehyde 16%	Electron Microscopy Sciences	15710
Penicillin/Streptomycin 100x	Life Technologies	15140122
Phosphate buffered saline (PBS) 1x	Corning	MT21031CV	Dulbecco's PBS (without calcium and magnesium)
Rainbow calibration particles, ultra kit	SpheroTech	UCRP-38-2K	3.5-3.9 µm, 2E6/mL
RPMI-1640 medium 1x	Life Technologies	11875-119
Sodium chloride 0.9% (USP)	Baxter Healthcare Corporation	2B1324
Software for cytometer data acquisition, "FACSDiva"	Becton Dickinson (BD)	n/a	Contact BD for license
Software for cytometer data analysis, "FlowJo"	TreeStar	n/a	Contact TreeStar for license
Trypsin-EDTA 0.25%	Life Technologies	25200-114